Assay for interferon

ABSTRACT

An assay for interferon comprises two antibodies to interferon, one labelled and at least one being a monoclonal antibody. The non-labelled antibody is suitably attached to a solid support such as polystyrene beads and the labelled antibody is suitably a radiolabelled monoclonal antibody, e.g. 125 I-NK2.

FIELD OF THE INVENTION

This invention relates to the field of immunometric assays. Inparticular it relates to an antibody excess immunometric assay forinterferon involving the use of monoclonal antibody to interferon.

BACKGROUND OF THE INVENTION

The interferons are a group of related proteins present in the mammalianbody. An interferon is a protein factor which exerts virus non-specific,antiviral activity at least in homologous cells through cellularmetabolic processes involving synthesis of both RNA and protein. Theinterferons are classified into types on the basis of antigenicspecificity, the designations being alpha, beta and gamma (thesecorrespond to previous designations leucocyte, fibroblast and type II(immune) interferons respectively). In addition to its antiviral effectinterferon has been implicated as a mediator or immune function of othercellular phenomena. Interferon research has been hampered by problems inits assay. The only widespread assays use tissue cultured cells andcompare some parameter of viral growth (for example viral RNA synthesisor host cell death) in the presence and absence of interferon. Thesecomplex biological assays, though sensitive, are laborious and subjectto inherent variability. In particular, components other than interferonpresent in the assay sample often influence viral growth. There is aneed for a simple indirect interferon assay. Such an assay would findwidespread application in at least three areas of interferon research:

(1) the monitoring of both laboratory and large-scale production andpurification of interferon;

(2) the quantitation of interferon doses in research and clinicalapplications;

(3) the measurement of interferon in biological fluids.

The object of the present invention is to provide an antibody excessimmuno assay for interferon which will fulfill this need.

The human body reacts to the presence of antigens by producing antibodymolecules from its lymphocyte cells. Antibodies have the property ofselectively binding to certain distinctive sites known as determinantson antibodies thereby rendering the antigen innocuous. The nature of theinteraction between antigen and antibody is not fully understood but itis clear that antibodies have a physical affinity for specificdeterminants of antigenic material. A reaction between an antibody and adeterminant on an antigen for which the antibody is specific results inan adduct, commonly referred to as an "immunocomplex". The formation ofsuch species makes possible a wide variety of assays for antigenicmaterial. Such assays are known generically as immuno assays.

Immunoassays fall broadly into two categories:

(1) Analyte excess; labelled antigen. (The term analyte is a term of artand in this context means "that to be analysed"). In this type of assayan antibody having specificity to the analyte is incubated with asolution containing the analyte and a known quantity of a labelledantigen. In this way a competitive equilibrium is set-up in which theunknown amount of analyte competes with the known amount of labelledantigen to form immunocomplexes with the antibody. A method ofdetermining the number of immunocomplexes formed between labelledantigen and the antibody make it possible to deduce the amount ofanalyte. This type of assay has certain disadvantages in that theultimate sensitivity of the assay is limited by the relative stabilityconstants of the immunocomplexes formed.

(2) Antibody excess; labelled antibody. In this type of assay theanalyte to be determined is incubated with an excess of labelledantibody molecules. The estimation of the amount of analyte is thereforelinear and its maximum sensitivity is, in theory at least, one moleculeof the analyte. A refinement of this method involves insolubilising anantibody to a solid substrate, in excess and allowing the analyte toform immunocomplexes therewith. Subsequently, an excess of labelledantibody to a second determinant on the analyte may be incubated withthe solid substrate. This type of assay, commonly referred to as a"sandwich assay", adds a great deal of specificity to the immuno assay.

The present invention is particularly concerned with the second type ofassay described, namely the antibody excess immunoassay, and isparticularly applied to an assay for interferon.

According to the present invention reagents for performing animmunoassay for interferon comprise two antibodies to interferon, atleast one of which is a monoclonal antibody, and one of which is alabelled antibody.

Conventional techniques for raising antibodies to interferon have provenproblematical, because of the extremely small quantities of pureinterferon available for immunisation in order to stimulate antibodyproduction in animals. The low antigenicity and small quantitiesavailable can only produce antiserum of moderate purity. Research in thefield of molecular biology has now provided an alternative source ofantibodies. It has been discovered that fusion between lymphocyte cellsand myeloma cells derived from mammals (for example, mice and rats) canproduce hybrid cells capable of replication in vitro (see Kohler andMilstein, Nature 256, 495 to 597). Such hybrid cells have the propertyof secreting an antibody of predefined specificity. This specificity isthat of the antibody produced by the lymphocyte involved in the fusion.The hybrid cells may be cloned and grown in stable culture to produce inthe culture supernatant samples of antibody to a specific determinant.Antibodies produced in this way are known as monoclonal antibodies inthe art.

The advantage of this technique is that it provides a source of aspecific antibody uncontaminated by antibodies raised to otherdeterminants either on the antigen with which the mammal was immunisedor on antigen impurities in the immunising material. Another advantageof the technique is that antigen not available in the pure form forscreening assays and present in the immunising material at lowconcentrations, for example interferon, may be used. Quite apart fromthe convenient source of antibody that the cell fusion techniquesprovides, the single determinant specificity of monoclonal antibodieshas great ramifications in the field of immunoassay. In particular amonoclonal antibody will bind only one determinant. The antibodiespreviously used in immunoassay, commonly known as polyclonal antibodies,do not have this specificity and assays using such polyclonal antibodieswere prone to inaccuracy as a result of this lack of specificity.

In one embodiment of the invention both antibodies to interferon aremonoclonal antibodies.

A particularly suitable monoclonal antibody to interferon is theHU--IFNα--specific monoclonal antibody NK2, the isolation and propertiesof which are described in a paper by D. S. Secher and D. C. Burke,Nature 285 at page 446 to 450 (1980).

As mentioned, both antibodies to interferon may be monoclonalantibodies. Conveniently however one of the antibodies is a monoclonalantibody, e.g. NK2, and the other is an antibody raised by conventionaltechniques. Such conventionally-raised antibodies may be raised fromhumans, sheep, horse, mouse, goat, guinea-pig, chicken, rat etc. Theywill normally be purified as far as appropriate and modified, e.g. byblocking, in known manner, to enhance their specificity.

The assay may be carried out in the liquid phase or with the use of asolid support. If it is a liquid assay it may be a homogeneous assay,wherein no separation of the reactants is necessary, or a heterogeneousassay, wherein separation of the reactants must take place. Separationmay be by means of an immunoprecipitation, by absorption by means ofcharcoal of the free antigen and antibody but not of the boundantigen-antibody complex, or by phase separation on the basis ofdifferent physical characteristics such as solubility.

The assay of the present invention is preferably carried out with theuse of a solid support, to which the non-labelled antibody is initiallyattached. The assay may thus be performed in several ways, namely (a)reaction of the interferon antigen with excess of the solid-phase linkedantibody followed by reaction of the product with the labelled antibody;(b) reaction of both the labelled antibody and the solid-phase linkedantibody together with the antigen; and (c) reaction of the labelledantibody with the antigen followed by reaction with an excess ofsolid-phase linked antibody.

The solid support may be fixed or free. Examples of fixed supports areplates, tubes, trays and wells. Examples of free supports are beads,particles and powders. Typical materials from which the supports may bemade include synthetic polymers, e.g. polystyrene, polyvinyl chloride,polyethylene, polyacrylamides, nylon and resins; natural polymers, e.g.cellulose, polysaccharides, sepharose, agarose, dextran; silica, glass,structural proteins such as collagen or polynucleotides, and cells, e.g.red blood cells, and Staphylococcus aureus. Attachment of the antibodyto the solid support may be by absorption, adsorption, or by a covalentlinkage, directly or by a linker.

The labelled monoclonal antibody may be isotopically or non-isotopicallylabelled. Preferably it is isotopically labelled, and the assay is thusan immunoradiometric assay (IRMA). The labelling may be direct orindirect (conjugate) and suitable labels include ¹²⁵ I, ¹³¹ I, ³² P, ¹⁴C and ³ H. Labelling techniques include the chloramine-T oxidationtechnique, the conjugation labelling technique (Bolton and Hunter,1973b, Biochem. J 133,529), and the PG,7 lactoperoxidase and iodogenprocedures.

Non-isotopic labels which may be used in the assay of the presentinvention include enzymes, and the assay may thus be an enzymeimmunoassay (EIA) or an enzyme linked immunosorbent assay (ELISA).Suitable enzyme markers include β-galactosidase, peroxidase, alkalinephosphatase, glucose oxidase etc. The assay may also be a fluorescentimmunoassay (FIA), examples of markers being fluorophores such asfluoroscein, rhodamine and chelated rare earths. The assay may be aluminescent immunoassay (LIA) involving bioluminescent orchemiluminescent markers, e.g. luminol.

Other forms of non-isotopic labelling involve cell tagging, the use ofheavy metals, co-enzymes, latex, free radicals and particle-counting(PACIA), all as known per se in the art.

The assay procedure is suitably conducted at temperatures in the range4° to 37° C., preferably, at room temperature. When it includes twosequential incubations, the first may continue for 4 hours or so, thesecond for 8 to 16 hours, e.g. overnight.

DESCRIPTION OF DIAGRAMS

FIG. 1 is a standard curve for measurement of interferon by immunoassay;

FIG. 2 is a standard curve for low levels of interferon;

FIG. 3 is a graph representing interferon assay in the presence of humanserum.

DETAILED DESCRIPTION OF THE EMBODIMENTS

As an example of a typical embodiment we describe an immunoradiometricassay (IRMA). In the assay to be described a sheep anti-interferonantibody is attached to a solid phase, polystyrene, and serves to anchorthe interferon present in the sample to the solid phase. The boundinterferon is then detected by the addition of ¹²⁵ I-NK2 (monoclonalanti-Hu-IFNα) and measurement of the counts bound to the solid phase.

The antibody NK2 was prepared as described by D. S. Secher and D. C.Burke in Nature, 285 at pages 446 to 450 (1980), and in Internationalpatent application No. PCT/GB 81/00067.

Three forms of polystyrene have been used as the solid phase.

(1) 3 ml test tubes (LP3, Luckham, Ltd., Bungers Hill, U.K.)

(2) 96-well microtiter trays (M24, Gibco Europe, Ltd. Uxbridge, U.K.)

(3) 6.5 mm beads (Northumbria Biologicals Ltd., Framlington, U.K.)

In the first case the whole tube was counted to measure the bound ¹²⁵I-NK2. When trays were used the bottom of each well was cut off with ahot wire and transformed to a clean tube for counting. In the third casethe beads were incubated in 20- or 60-well trays (93-0402, AbbottLaboratories, Basingstoke, U.K.) and transferred to tubes for counting.The incubation volumes used were 1 ml or 0.1 ml (wells) and 0.2 ml(beads). All three supports were found to be satisfactory and forassaying large numbers of samples the beads were preferred for theirconvenience.

NK2 antibody was purified from the serum and ascites fluid of micecarrying NK2 tumours by ammonium sulphate precipitation and DEAEion-exchange chromatograph, labelled by the chloramine-T method anddesalted on Sephadex G-50 (fine) column. The labelled IgG had a specificactivity of about 2 Ci/μ mole or about 1 atom ¹²⁵ I per molecule IgG.

In each assay a standard curve was constructed using either theinterferon reference standard MRC 69/19 or a laboratory standard.

In one such assay (see FIG. 1) IgG was purified from a sheepanti-Hu-IFNαantiserum (450,000 neutralising units/ml) by ammoniumsulphate precipitation and DEAE-cellulose ion exchange chromatographyand coated on to polystyrene beads by incubation of the beads at 4° C.for 16 hrs. in the sheep IgG (20 μg/ml in PBS--phosphate buffersolution--, 5 mM EDTA, 0.1% NaN₃). Several hundred beads were coated,washed in a medium (HS medium) consisting of PBS, 10% horse serum, 0.1%NaN₃ and stored in HS medium at 4°. Assay trays (20- or 60-well, Abbott)were incubated with HS medium at 4° to block any sites for proteinattachment. Interferon samples were diluted in HS medium and duplicatesamples (200 μl) added to antibody-coated beads. After 4 h at 4° thebeads were washed with 12 ml HS medium each using a combineddispenser/aspirator ("Pentwash", Abbott Laboratories). After removal ofany residual medium, ¹²⁵ I-NK2 (purified IgG, 40,000 cpm) was added andincubated for 16 h at 4°. The beads were washed as before andtransferred to a gamma-counter. The broken line indicates the backgroundcpm bound when the coated beads were incubated in HS medium withoutinterferon. The counts bound at 8,000 U/ml were at a maximum; no furtherincrease was observed with interferon concentrations up to 10⁶ U/ml. Thelow level of non-specific binding (<1% of input counts is an importantcharacteristic of the sandwich assay).

In a second experiment (see FIG. 2) we showed that at low interferonconcentrations the counts bound are proportional to the interferonconcentration. Interferon samples were prepared by dilution of thereference research standard in HS medium and assayed as described forFIG. 1 except that only 23,000 cpm ¹²⁵ I-NK2 was added to each bead. Thedotted line indicates the background cpm bound and was the mean of eightvalues (s.e.=±6 cpm). It can be seen that concentrations of ≧50 U/ml canbe easily measured. The standard assay conditions (i.e. those used inthe construction of FIG. 1) have been adjusted for the convenientmeasurement of samples encountered in the purification of interferon.Such samples have values of 10³ -10⁷ U/ml. Solutions containing 10³ U/mlare diluted by serial dilution and the titration curve obtained matchedto the standard curve. Comparison of the above immunoassay techniquewith the known antiviral assay technique on the same samples shows goodagreement, with experimental error.

The immunoassay of the present invention offers considerable advantagesover the conventional biological assay. Since the antibody-coatedpolystyrene can be used at 4°, samples can be assayed at short noticeand the results are obtained within 24 hours of beginning the assay. Thestandard curves show very little variation from assay to assay comparedto the much greater inherent variability of biological assays. Fourmeasurements of a solution of 2000 U/ml gave a value of 3063±345 cpm inindependent assays in which the input cpm ranged from 47000 to 53000cpm. Only small amounts of sheep antibody are necessary, since the IgGsolution used to coat the polystyrene can be re-used without apparentlyreducing the sensitivity of the assay. The quality of the sheep antibodyis not a critical factor in the success of the assay, and otherantibodies to Hu-IFNα, as described above, may substitute equally well.The assay exploits the specificity of the monoclonal antibody NK2 andsince this is the product of a hybrid myeloma cell line in culture itcan be produced in large amounts without any change in quality. Finallythe assay is inexpensive (especially since all the plastic ware exceptthe beads may be re-used), lends itself to automation and it is possiblefor one person to assay several hundred samples in a day.

A limitation in the conventional assay of interferon in serum or otherbiological fluids has been the need to dilute the sample until theeffects of other non-interferon substances that affect viral growth nolonger mask the action of the interferon. Dilution of a sample ofinterferon purified by affinity chromatography on NK2-Sepharose ineither PBS, 10% horse serum, 0.1% NaN₃ or in undiluted normal humanserum resulted in the identical titration curves shown in FIG. 3indicating that the immunoassay of the invention overcomes this problem.In this experiment interferon was (a) diluted in HS medium or (b)undiluted normal human serum and samples assayed as described inrelation to FIG. 1 except that 3 ml test tubes were used as thepolystyrene support rather than the beads. The solid line represents thecpm bound in case (a) and the broken line the results in case (b).

The lower limit in the measurement of serum interferon by biologicalassays has been sufficient to detect interferon in the serum of somepatients to whom interferon has been administered. This sensitivity isinsufficient, however, to measure the interferon concentration (if any)in normal human serum.

When conditions are adjusted to measure concentrations of interferon asdescribed above the immunoassay of the invention can reliably measurearound 50 U/ml. The sensitivity of the assay can moreover be furtherincreased by prior concentration and purification of the interferon on asmall NK-2-affinity column.

I claim:
 1. An immunoassay for interferon involving an interferonantibody attached to a solid support, and a labelled monoclonalantibody, each of which antibodies is capable of specifically binding toat least one antigenic determinant of interferon, said processcomprising the steps of:placing a sample to be assayed in contact withthe solid support allowing immunocomplexes to form between interferon inthe sample and the interferon antibody bound to the solid support,placing the labelled monoclonal antibody in contact with the solidsupport allowing immunocomplexes to form between interferon bound to thesolid and the labelled monoclonal antibody, and estimating theimmunocomplexes.
 2. An immunoassay for interferon involving aninterferon antibody attached to a solid support, and a labelledmonoclonal antibody, each of which antibodies is capable of specificallybinding to at least one antigenic determinant of interferon, saidprocess comprising the steps of:reacting the labelled monoclonalantibody with the sample to be assayed, reacting the reaction productwith an excess of the solid support linked antibody, and estimating theimmunocomplexes formed.
 3. An immunoassay for interferon involving aninterferon antibody attached to a solid support, and a labelledmonoclonal antibody, each of which antibodies is capable of specificallybinding to at least one antigenic determinant of interferon, saidprocess comprising the steps of:reacting the labelled monoclonalantibody and the solid support linked antibody with the sample to beassayed, and estimating the immunocomplexes formed.
 4. An immunoassay asclaimed in claim 1, wherein the labelled monoclonal antibody is derivedfrom the NK2 cell line.
 5. An immunoassay as claimed in claim 2, whereinthe labelled monoclonal antibody is derived from the NK2 cell line. 6.An immunoassay as claimed in claim 3, wherein the labelled monoclonalantibody is derived from the NK2 cell line.
 7. An immunoassay reagentkit for use in the assay of interferon comprising:a first containercontaining a first antibody, and a second container containing a secondantibody, each of which antibodies is capable of specifically binding toan antigenic determinant of interferon, at least one of which antibodiesis a monoclonal antibody and one of which antibodies is a labelledantibody.
 8. A kit as claimed in claim 7 wherein the labelled antibodyis a monoclonal antibody capable of specifically binding to adeterminant of human interferon-α.
 9. A kit as claimed in claim 8wherein the monoclonal antibody is derived from the NK2 cell line.
 10. Akit as claimed in claim 9 wherein the monoclonal antibody is aradio-labelled monoclonal antibody derived from the NK2 cell line.
 11. Akit as claimed in claim 7 wherein both antibodies are monoclonalantibodies, one of which is labelled.
 12. A kit as claimed in claim 7wherein a non-labelled antibody is bound to a solid support.
 13. Animmunoassay reagent for use in the assay of interferon comprisinga firstantibody, and a second antibody each of which antibodies is capable ofspecifically binding to an antigenic determinant of interferon, at leastone of which antibodies is a monoclonal antibody and one of whichantibodies is a labelled antibody.
 14. A reagent as claimed in claim 13wherein the labelled antibody is a monoclonal antibody capable ofspecifically binding to a determinant of human interferon-α.
 15. Areagent as claimed in claim 14 wherein the monoclonal antibody isderived from the NK2 cell line.
 16. A reagent as claimed in claim 15wherein the monoclonal antibody is a radio-labelled monoclonal antibodyderived from the NK2 cell line.
 17. A reagent as claimed in claim 13wherein both antibodies are monoclonal antibodies, one of which islabelled.
 18. A reagent as claimed in claim 13 wherein the non-labelledantibody is bound to a solid support.